HCV is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 9.5 kb that encodes about 3010 amino acids (Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Takamizawa et al., J. Virol. (1991) 65:1105-1113). The HCV polyprotein is processed by host and viral proteases into several mature proteins: core protein (C), envelope glycoproteins (E1 and E2), and six nonstructural proteins (NS2, NS3, NS4a, NS4b, NS5a, NS5b) (Hijikata et al. Proc. Natl. Acad. Sci. USA (1993) 90:10773-10777; Grakoui et al., J. Virol. (1993) 67:1385-1395). NS3 is a 630 amino acid protein with three enzymatic activities: the approximately N-terminal 180 amino acids provide serine protease function whereas the remaining C-terminal domains have both helicase and NTPase activities (Bartenschlager et al., J. Virol. (1993) 67:3835-3844; Kim et al., Biochem. Biophys. Res. Commun. (1995) 215:160-166; Preugschat et al., J. Biol. Chem. (1996) 271:24449-24457). The NS3 protease is responsible for cleavages at the NS3/4a, NS4a/4b, NS4b/5a and NS5a/5b junction sites (Grakoui et al., J. Virol. (1993) 67:2832-2843). NS4a includes approximately 54 amino acids and acts as a cofactor of the NS3 protease and is essential for polyprotein processing (Failla et al., J. Virol. (1994) 68:3753-3760).
HCV is the major etiologic agent for blood transfusion-associated and community-acquired non-A, non-B viral hepatitis (Alter et al., N. Engl. J. Med. (1989) 321:1494-1500; Choo et al., Science (1989) 244:359-362; Kuo et al., Science (1989) 244:362-364). HCV currently affects approximately 3% of the world's population. 70% of these individuals develop HCV chronic infection, which often progresses to liver cirrhosis and hepatocellular carcinomas (Bruix et al., Lancet (1989) 2:1004-1006; Saito et al., Proc. Natl. Acad. Sci. USA (1990) 87:6547-6549). The incidence of posttransfusion HCV has steadily declined since the implementation of routine screening for HCV antibodies among blood donors (Pillonel et al., Transfusion (2002) 42:980-988). Despite the proven utility of these assays for blood screening and for the diagnosis of HCV infection in symptomatic patients, important challenges to the improvement of assay performance remain. Such challenges include reducing the window of seronegativity, improving the detection of HCV samples from immunosuppressed patients, and increasing assay sensitivity to detect antibodies to the different HCV genotype-specific epitopes.
Several assays have been developed for the serodiagnosis of HCV infection. See, e.g., Choo et al., Science (1989) 244:359-362; Kuo et al., Science (1989) 244:362-364; Choo et al., Br. Med. Bull. (1990) 46:423-441; Ebeling et al., Lancet (1990) 335:982-983; van der Poel et al., Lancet (1990) 335:558-560; van der Poel et al., Lancet (1991) 337:317-319; Chien, D. Y., International Publication No. WO 94/01778; Valenzuela et al., International Publication No. WO 97/44469; and Kashiwakuma et al., U.S. Pat. No. 5,871,904.
The current commercially licensed HCV ELISA antibody tests mainly use recombinant proteins containing linear epitopes. For example, three recombinant HCV proteins from core (C22-3), NS3 and NS4 regions (C200) and NS5 are used in one of the commercially available HCV assays (Uyttendaele et al., Vox Sang. (1994) 66:122-129).
U.S. Pat. No. 6,632,601, incorporated herein by reference in its entirety, describes immunoassays using NS3/4a conformational epitopes, in combination with multiple epitope fusion antigens (MEFAs). For a description of HCV MEFAs see, e.g., Chien et al., J. Clin. Microbiol. (1999) 37:1393-1397; International Publication No. WO 97/44469; U.S. Pat. Nos. 6,514,731, 6,428,792; 6,632,601; and U.S. Patent Publication No. 20040142321. The assays using these reagents provide sensitive and reliable methods for detecting early HCV seroconversion. NS3/4a, expressed in yeast and purified under non-denaturing conditions as described in U.S. Pat. No. 6,632,601, contains both protease and helicase function. Because NS3/4a purified in this manner preserves the native conformation, it has been found to be more sensitive than the c200 or c33c antigens in early seroconversion antibody detection. In antibody assays using NS3/4a and MEFA 7.1 as antigens, seroconversion antibodies were detected 2-14 days earlier than currently marketed HCV assays. However, the NS3/4 protein undergoes self-hydrolysis and also cleaves MEFA 7.1 due to the NS3 protease activity.
International Publication Nos. WO 04/00547 and WO 01/38360, and commonly owned Provisional Patent Application No. 60/604,858, describe HCV proteins including mutated NS3 protease domains with reduced proteolytic activity. However, there remains a need for sensitive, accurate diagnostic and prognostic tools in order to provide adequate patient care as well as to prevent transmission of HCV by blood and blood products or by close personal contact.